3D Immuno-DNA Fluorescence In Situ Hybridization (FISH) for Detection of HIV-1 and Cellular Genes in Primary CD4⁺ T Cells

Abstract

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture–based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.

In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4⁺ T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.